Non-human mammal mutant for expression of the alpha6 subunit of the nicotinic acetylcholine receptor, and use for screening substances that can interact with said receptor

ABSTRACT

The invention concerns a non-human mammal carrying a mutation in the gene coding for the alpha6 subunit of the nicotinic acetylcholine receptor (nAChR), said mutation preventing expression of said nAChR alpha6 subunit in a functional form in the mammal.  
     The invention also concerns synaptosome preparations obtained from said animals and cell cultures obtained by mutation of the alpha6 subunit as defined above.

[0001] The present application relates to non-human mammals mutant for expression of the alpha6 subunit of the nicotinic acetylcholine receptor, hereinafter abbreviated to “nAChR”, and to the use of said mammals for identifying or screening for molecules that can interact with said receptor.

[0002] Little is currently known about the role of nicotinic acetylcholine receptors in the central nervous system. Such receptors (Marubio, L. M., et al. Reduced antinociception in mice lacking neuronal nicotinic receptor subunits. Nature, 1999. 398: p. 805-810) are involved in nociception, memory, locomotion control, nicotine dependence, and certain neurodegenerative disorders such as Parkinson's disease or Alzheimer's disease (Newhouse, P., et al. Nicotinic system involvement in Alzheimer's and Parkinson's diseases. Implications for therapeutics. Drugs Aging, 1997. 11 (3): p 206-228), but the mechanisms of that involvement have not been identified.

[0003] Regarding nicotine dependence, recent works have shown the importance of the beta2 subunit of nAChR as regards nicotine dependence properties in rodents (Picciotto, M. et al. Acetylcholine receptors containing beta2-subunit are involved in the reinforcing properties of nicotine. Nature, 1998 391: p. 173-177). In that prior works, mice lacking the beta2 subunit were incapable of maintaining auto-administration behaviour when cocaine was replaced by nicotine. That study also underlined the absence of an electrophysiological response of the mesencephalic dopaminergic neurons of mutant mice in response to nicotine. Furthermore, microdialysis experiments carried out on mutant mice showed that the release of dopamine was affected in the nucleus accumbens (corresponding to the ventral portion of the striatum) after systemic administration of nicotine. Those observations contributed to highlighting the parallel that may exist between nicotine and other addictive drugs such as heroin or cocaine in which the dependence effects are linked to an increase in the basal level of dopamine in the nucleus accumbens.

[0004] However, the extended cellular expression profile of the beta2 subunit did not allow an accurate definition of the regions of the brain contributing to initiation of nicotine dependence, and the nature of the alpha subunits associated with the beta2 subunit in that process were not identified.

[0005] Of the eight alpha subunits of nAChR already identified in the brain of rodents, certain, in particular the alpha4 subunit, have been used to examine their properties.

[0006] In the present application, the inventors have chosen to concentrate on a particular subunit, the alpha6 subunit of neuronal nicotinic acetylcholine receptors, this subunit possibly being associated with the beta2 subunit and having an expression profile restricted to catecholaminergic neurons (Le Novere, N. et al. Neuronal nicotinic receptor alpha6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain, European Journal of Neuroscience, 1996, 8(11): p. 2428-39). In a highly advantageous manner in the present application, the inventors have selected this subunit for its expression profile that is principally localized to the substantia nigra, the ventral tegmental area and the locus coeruleus.

[0007] Studies (Le Novere, N. et al. Involvement of alpha6 nicotinic receptor subunit in nicotine-elicited locomotion, demonstrated by in vivo antisense oligonucleotides infusion. NeuroReport, 1999. 10: p. 2497-2501) have shown that antisense oligonucleotides can demonstrate the importance of the alpha6 subunit in the hyperlocomotive effect of nicotine in the usual environment, an effect which would be mediated by the mesencephalic dopaminergic system. Those elements have led certain authors to hypothesize that the alpha6 subunit could play a role in regulating the release of dopamine by nicotinic agonists (Reuben M. B. S. et al. Nicotinic receptors modulating somatodendritic and terminal dopamine release differ pharmacologically. Eur. J. Pharmacol, 2000, 393(1-3): p. 39-49). However, those authors have observed that the result obtained on the potential and efficacy of the test agonist associated with dopamine release does not resemble that which was obtained with recombinant nictonic cholinoreceptors containing the alpha6 subunit.

[0008] The invention provides means that allow in vivo observation or observation in an in vitro system that can approach the existing in vivo conditions, to identify or screen for molecules that can interact in a ligand/receptor type reaction with nicotinic acetylcholine receptors and in particular with those comprising the alpha6 subunit of said receptors.

[0009] Thus, the invention concerns non-human mammals, in particular rodents and more particularly mice, which are mutant for the gene coding for the nAChR alpha6 subunit, said mammals being depleted in functional alpha6 subunit. In the remainder of the description, these animals will be termed “knockout” to qualify the mutation that they have in the gene coding for the alpha6 subunit of nAChR receptors. These mammals can constitute animal models for identifying substances, in particular substances having pharmacological properties, which interact with said alpha6 subunit in the nAChR receptors. These receptors comprising the alpha6 subunit can be of type 2α6, 2β2, β3 (Le Novere N. et al. European Journal of Neuroscience, 1996, 8(11), p 2428-39).

[0010] In particular, by dint of these animals or products, in particular cell cultures which can be derived therefrom, the invention provides a model that allows to determine the selectivity of substances of interest towards nAChR receptors present in certain neuronal populations and, in particular in the catecholaminergic neurons.

[0011] Given substances can be tested on animals expressing the nAChR alpha6 subunit normally and the same test can be carried out on animals in accordance with the invention that are mutant for this subunit expressed in the functional form, to allow the respective effects observed to be compared, in particular any interaction of the test substance with nicotinic transmission and dopaminergic transmission mediated by the alpha6 subunit of nAChR receptors.

[0012] The means of the invention are particularly suitable to screening and in particular to pharmacological screening of substances that may behave as ligands for the nAChR receptors, via their interaction with the nicotine binding site of the alpha6 subunit, and in particular capable of a nicotinic type agonist or antagonist activity. Particularly advantageously, the identified substances bind to nAChRs via the alpha6 subunit and have a restricted specificity for dopaminergic neurons. This specificity can be detected because of expression of the nAChR alpha6 subunit, restricted to those dopaminergic neurons.

[0013] As an example, it has been observed that Parkinson's disease is characterized by a selective degeneration of dopaminergic neurons of the substantia nigra.

[0014] The capacity of the identified substances to behave as ligands for the nAChR alpha6 subunit defines, within the context of the invention, substances having an activity comparable to that of acetylcholine, or to that of agonists such as nicotine, at least as regards the capacity of acetylcholine or nicotine to bind to nAChR receptors comprising the alpha6 subunit. Examples of substances having such properties are nicotine, cytisine, epibatidin, and their derivatives obtained by chemical modification.

[0015] Examples of antagonist substances binding to said nAChR alpha6 subunit are DhβE, hexamethotium, alpha-bungarotoxin and n-bungarotoxin. Examples are mentioned in the publication by Spang J. et al. (Chem Biol, 2000 7(7): p 545-555) and Sharples C. et al. (J Neurosci, 2000, 20(8): p 2783-91).

[0016] Available results on the neuroprotective effect of nicotine in Parkinson's disease (Newhouse P. et al. Drugs Aging, 1997, 11(3): p 206-228) open the doors to new possibilities for different therapies with nicotinic agents. The design of nicotinic substances, which may have therapeutic properties and may be used in the design of a drug, which may have a selectivity for receptors containing the alpha6 subunit, could be of advantage in particular as regards avoiding undesirable side effects such as the cardiovascular risks associated with exposure to nicotine.

[0017] Thus, the invention provides a non-human mammal carrying a mutation in the gene coding for the alpha6 subunit of the nicotinic acetylcholine receptor (nAChR), said mutation preventing expression in the mammal of said nAChR alpha6 subunit in a functional form.

[0018] The expression “functional form” as used in the context of the invention means the form of the alpha6 subunit (α6) of nAChR which allows recognition and interaction between this subunit and agonists or antagonists of receptors in which it is present, and more particularly which allows binding to acetylcholine. The functional form of the alpha6 subunit is thus that in which said subunit comprises a functional binding site for agonist or antagonist molecules.

[0019] The functional nature of the binding site can be verified by the fact that acetylcholine, present or administered under conditions allowing its interaction with nAChr receptors comprising the alpha6 subunit, modifies the amount of dopamine release compared with the amount of dopamine which can be released when the alpha6 subunit is absent or non-functional. In other words, the absence of the alpha6 subunit or its presence in a non functional form alters the amplitude and the pharmacology of the response of nAChR receptors to nicotine. The examples describe the tests that can be used in this regard.

[0020] The remainder of the text will also refer to the “functional form” of the gene to translate the capacity of the gene to express an alpha6 subunit in the functional form. The non-human mammals of the invention are thus those which have undergone genetical inactivation of the gene coding for the alpha6 subunit of nAChR.

[0021] In a first implementation of the invention, the non-human mammal is characterized in that the gene coding for the nAChR alpha6 subunit is mutated by deletion of all or a portion of its exons.

[0022] A binding site for the nAChR alpha6 subunit recognized by nicotine and by other agonists and antagonists of this receptor is coded by exons 2, 3 and 4 of the gene coding for said alpha6 subunit.

[0023] Further, deletion of exons 1 and 2 by their destruction prevents correct translation of mRNA (because ATG and splicing sites are destroyed) even in the gene transcription hypothesis. In fact, the absence of exons 1 and 2 prevents gene transcription.

[0024] Thus, the invention particularly pertains to a non-human mammal, characterized in that the gene coding for the nAChR alpha6 subunit is mutated by deletion of at least a portion of exon 1 and/or at least a portion of exon 2, which, in the nAChR alpha6 subunit expressed in the mammal, is sufficient to prevent binding of said nAChR alpha6 subunit with its agonists and/or antagonists.

[0025] Preferably, the non-human mammal of the invention is mutated by essentially complete deletion of exons 1 and 2.

[0026] In particular, the invention pertains to a non-human mammal, in particular a rodent and especially a mouse, characterized in that the mutation of the gene coding for the nAChR alpha6 subunit is homozygous in nature.

[0027] The mutation is, for example, such that the mammal has phenotype (α6^(−/−)) and no longer expresses the nAChR alpha6 subunit, the two alleles of the gene being mutated in similar manner.

[0028] In a further implementation, the invention concerns a non-human mammal characterized in that the mutation for the gene coding for the nAChR alpha6 subunit is homozygous, said mammal having a (α6^(Δ-2/Δ1-2)) phenotype.

[0029] Phenotype (α6^(Δ1-2/Δ1-2)) corresponds to expression of a gene coding for the alpha6 subunit, said gene being truncated at exons 1 and 2, so that said subunit is effectively expressed and in particular comprises the transmembrane portion of the native alpha6 subunit; the expressed receptor is not functional for binding agonists which is normally dependent on correct transcription and translation of exons 1 and 2.

[0030] Said non-human mammals can be obtained by the known technique of the skilled, of homologous recombination of the wild type gene coding for the nAChR alpha6 subunit, with a construct of the type of the target construct of FIG. 1. The homozygous nature of the mutation can result from crossing two animals with one mutated gene allele, the homozygous descendants then being selected. Alternatively, the homozygous nature of the mutation can be obtained directly from ES cells by increasing the concentration of neomycin used for selection and retaining only those clones that have undergone homologous double recombination.

[0031] The non-human mammals of the invention can be obtained so that non functional expression of the nAChR alpha6 subunit or the absence of expression of this alpha6 subunit is constitutive, the animal being mutated for the functional gene or incapable of expressing its transcription product.

[0032] Alternatively, the invention concerns non-human mammals, characterized in that the mutation of the gene coding for the nAChR alpha6 subunit is of the inducible or conditional type.

[0033] The inducible or conditional nature of the mutation can be useful in particular because the animal which is mutated by induction for expression of the gene for the nAChR alpha6 subunit in the functional form is not in a position to undergo behavioral adaptations, for example by compensation, and thus of modifying its behaviour towards agonists or antagonists, and in general towards any substance that can interact normally with the native nAChR alpha6 subunit.

[0034] Mammals in accordance with the invention in which the mutation in question is inducible can be obtained by specific recombination techniques allowing recombination intended to establish deletion or mutation of a gene to be carried out under the control of certain agents, in particular tetracycline or RU486 (Kellendonk C. et al. J. Mol. Biol. 1999, 285(1): p 175-82), for example in certain neuronal cell populations.

[0035] Inducible gene inactivation lies in using an inducible promoter (for example the TRE/rtta system (Gossen M. et al. Science, 1995, 268: p 1766-9)) which controls expression of an enzyme, Cre-recombinase. In that system, transgenic animals are generated that express both the synthetic activator rtta under the control of a specific promoter in certain tissues, and Cre-recombinase under the control of the inducible promoter TRE. In the presence of tetracycline, the rtta activator binds to the TRE promoter and induces expression of Cre-recombinase only in the cells in which rtta is expressed. In parallel, homologous recombination produces animals in which the gene coding for the alpha6 subunit is flanked by two LoxP sites. These sites are the target for Cre-recombinase which causes excision of the entire DNA fragment located between two LoxP sites. By crossing these animals, descendants are obtained in which excision of the alpha6 gene can be induced simply by adding tetracycline to the drinking water.

[0036] The LoxP sites in question can alternatively be inserted into the gene to frame a fragment of the gene, deletion of which is sufficient to inactivate the gene or lead to expression of a non-functional alpha6 subunit.

[0037] Other equivalent systems can be used.

[0038] The non-human mammals of the invention, in particular rodents and especially mice, can be obtained by crossing animals carrying the genetic constructions necessary for producing the desired mutation of the gene coding for the nAChR alpha6 subunit and in a manner so as to render said mutation homozygous. The invention also concerns animals descended from said animals.

[0039] The invention also concerns knockout non-human mammals in which the gene for the nAChR alpha6 subunit is heterozygously mutated. Because of this heterozygous mutation, said animals have, a reduced sensitivity to ligands for the alpha6 subunit and are thus of interest in confirming that an observed reduction in an agonist or antagonist effect towards the alpha6 subunit with a substance can be attributed to the mutation.

[0040] Different constructs have been produced to render preparation of the animals of the invention possible. These constructs are described in detail in the following examples and in particular, the invention concerns a non-human mammal and in particular a mouse, characterized in that its gene coding for the nAChR alpha6 subunit is mutated by homologous recombination with the sequence designated a6KO contained in the E. coli strain deposited at the CNCM with accession number 1-2550 on Aug. 29, 2000.

[0041] The invention also concerns cell populations and in particular cell cultures as obtained from a non-human mammal as described above.

[0042] Within the context of the invention, these cell populations are neuronal populations and in particular cell populations that normally express the alpha6 subunit of nAChR receptor, such as catecholaminergic neurons. Examples of advantageous cell populations are mesencephalic neuronal cells.

[0043] The invention also concerns preparations of striatal synaptosomes.

[0044] The invention also concerns the use of a non-human mammal, a synaptosome preparation or a cell culture as defined as above for the study of the effects of given substances on nicotinic receptors.

[0045] One particularly advantageous use of the invention in this context is for the study of the effects of addictive drugs on the nicotinic receptors of acetylcholine, said drugs possibly already being known and identified as such in general or being known without their properties having been identified, or being novel as regards their structure and functions.

[0046] In particular, the use of animals of the invention or synaptosome preparations or derived cell cultures allows, for example by measuring the amount of dopamine release, the effect of agonists for the nicotinic acetylcholine receptors (nAChR) in particular the alpha6 subunit of these receptors, to be determined.

[0047] Particularly advantageously, the animals, synaptosome preparations, or cell cultures of the invention can be used in screening protocols, in particular pharmacological protocols, for nAChR agonist or antagonist substances.

[0048] The specificity of a test substance towards a nAChR receptor can be characterized by observation, for a substance, of a high activity (i.e., an activity of the order of that obtained with nicotine, or higher) on a mouse or other wild type mammal and the absence of activity on a mouse or other mammal in accordance with the invention.

[0049] More specifically, the knockout animals of the invention or the cell cultures or the synaptosome preparations allow identification, from agonists or antagonists for the nicotinic acetylcholine receptors, of those which can have a restricted effect to catecholaminergic neurons, in particular restricted to dopaminergic neurons.

[0050] The test substances are known substances, for example known for their agonist or antagonist properties as regards receptors and in particular as regards nAChR receptors. They can also be novel substances which the invention can be used to identify certain properties thereof, in particular certain pharmacological characteristics as regards the nAChR receptors.

[0051] The invention concerns a method for screening substances that are capable of interacting with the nAChR alpha6 subunit, comprising:

[0052] bringing the selected substance into contact with a non-human mammal of the invention, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors;

[0053] detecting the ligand/receptor type interaction.

[0054] Thus, the invention provides a method for screening substances that can interact with the nAChR alpha6 subunit, selected from substances interacting with nAChR receptors, comprising:

[0055] bringing the selected substance into contact with a non-human mammal of the invention, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors;

[0056] detecting the amount of dopamine released in the striatum.

[0057] The invention also concerns a method for screening substances that can interact with the nAChR alpha6 subunit, selected from substances interacting with nAChR receptors, comprising:

[0058] bringing the selected substance into contact with a cell culture in accordance with the invention, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors;

[0059] detecting the amount of dopamine released.

[0060] The invention also concerns a method for screening substances that can interact with the nAChR alpha6 subunit, selected from substances interacting with nAChR receptors, comprising:

[0061] bringing the selected substance into contact with a synaptosome preparation in accordance with the invention, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors;

[0062] detecting the amount of dopamine released.

[0063] Examples of other methods for detecting the interaction of the screened substances with the nAChR alpha6 subunit are those which employ a double hybrid system such as those described in International patent application WO-99/42612.

[0064] The variation in the detected dopamine level can be evaluated by comparison of the basal level of dopamine released in an animal expressing the nAChR alpha6 subunit normally, or released in a synaptosome preparation carried out from such an animal, or in a cell culture expressing this subunit, with the level of dopamine released when the animal, preparation or culture in question is brought into contact with the test substance under conditions that allow its interaction with the alpha6 subunit.

[0065] In a further mode of the invention, the screening methods employ substances the capacity of which to bind to nAChRs has not previously been studied.

[0066] In a further implementation, the cell cultures of the invention are used to study or identify, from agonist or antagonist substances for the nicotinic acetylcholine receptors, those which have a neuroprotective effect.

[0067] Thus, the invention provides a method for screening substances that can interact with the nAChR alpha6 subunit, selected from substances interacting with nAChRs, comprising:

[0068] bringing the selected substance into contact with a cell culture in accordance with the invention, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors;

[0069] apply a stress to said cell culture;

[0070] observing the neuroprotective effect of said substance on said culture as regards said stress.

[0071] Stressing a cell culture has been described, for example in the publication by Akaike A et al., (Brain Res 1994, 644(2): p 181-7). The effect of said stress may be cell death. In this case, observation of the neuroprotective effect may consist of comparing the cell death caused by this stress on a culture expressing the alpha6 subunit with that caused by said stress on the cell culture of the invention, said cell cultures having previously been brought into contact with the screened substance.

[0072] The method of the invention as defined, which can be implemented for the uses described above in accordance with the invention, allows substances having an affinity for the alpha6 subunit to be identified, and which interact with nicotinic transmission and dopaminergic transmission in a manner that is selective by binding to receptors comprising the alpha6 subunit via said subunit. This method is of particular advantage when studying the interaction of addictive drugs with the alpha6 subunit of nAChR and in particular when studying their selectivity towards identified neuronal populations.

[0073] The possible variation in the amount of dopamine released in the striatum of an animal of the invention can be detected using known techniques, in particular the techniques described in the examples below.

[0074] As an example, after implementing the method of the invention, the variation, in particular increase, will be detected in the level of dopamine released compared with the base dopamine level detected in an animal expressing the nAChR alpha6 subunit normally.

[0075] When the mutation in the alpha6 subunit is conditional or inducible, the desired basal level of released dopamine can be detected prior to inducing the mutation and subsequently compared with the dopamine level in the same animal in which the mutation has been induced.

[0076] In that case, the level of released dopamine is measured by microdialysis (Marshall D. et al. J. Neurochem, 1997, 68(4): p 1511-9) In a preferred implementation of the method of the invention, the test substances are substances for which the selective affinity for nicotinic acetylcholine receptors restricted to dopaminergic neurons is to be determined.

[0077] In a preferred implementation of the method of the invention, the test substances are substances having an affinity for nAChR receptors comprising an alpha6 subunit for which the selectivity towards this subunit is sought, the method comprising:

[0078] a) bringing the selected substance into contact with a non-human mammal as hereinbefore defined, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors;

[0079] b) detecting the dopamine released in the striatum;

[0080] c) comparing the amount of dopamine released in the striatum in step b) with the amount of dopamine released detected in the striatum of the non-human mammal expressing the nAChR alpha6 subunit.

[0081] In a further implementation, this method comprises:

[0082] a) bringing the selected substance into contact with a synaptosome preparation as defined in the invention, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors;

[0083] b) detecting the amount of dopamine released;

[0084] c) comparing the amount of dopamine released detected in step b) with the amount of dopamine released in a synaptosome preparation from a non-human mammal expressing the nAChR alpha6 subunit.

[0085] In a further implementation, this method comprises:

[0086] a) bringing the selected substance into contact with a cell culture as defined in the invention, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors;

[0087] b) detecting the amount of dopamine released;

[0088] c) comparing the amount of dopamine released detected in step b) with the amount of dopamine released in a cell culture expressing the nAChR alpha6 subunit.

[0089] Steps b) and c) of said methods can be replaced by any step allowing detection of the ligand/receptor interaction.

[0090] In a particular implementation of the screening methods of the invention, the non-human mammals, synaptosome preparations or cell cultures are controls for the interaction of the selected substances with the alpha6 subunit of nAChR receptors and these methods comprise a step in which the effect of the test substance on the alpha6 subunit is determined on an animal that is wild type for expression of said alpha6 subunit or on a preparation of synaptosomes of such an animal or on a cell culture expressing said subunit normally.

[0091] The invention also concerns the substances identified at the end of a screening method in accordance with the invention, and their use in the preparation of compositions, in particular for therapeutic purposes.

[0092] The invention also concerns vectors, and in particular plasmids comprising all or a portion of a gene coding for the alpha6 subunit of nAChR receptors, in particular vectors carrying a mutated gene for the alpha6 subunit, said vectors possibly constituting means for preparing constructions intended to produce the animals of the invention.

[0093] To this end, particularly advantageous plasmids are those selected from the following plasmids deposited at the CNCM (Collection National de Culture de Microorganismes, Paris, France):

[0094] a6HN: CNCM N° 1-2500 deposited on Jun. 22, 2000;

[0095] a6EH: CNCM N° 1-2501 deposited on Jun. 22, 2000;

[0096] a6EE: CNCM N° 1-2502 deposited on Jun. 22, 2000;

[0097] a6PP: CNCM N° 1-2503 deposited on Jun. 22, 2000;

[0098] a6KO: CNCM N° 1-2550 deposited on Aug. 29, 2000;

[0099] or the bacterial artificial chromosome (BAC) designated a6BAC deposited at the CNCM with accession number 1-2504 on Jun. 22, 2000.

[0100] The invention also concerns a sequence of nucleotides contained in one of the plasmids identified above or in the bacterial artificial chromosome also identified above.

[0101] A preferred sequence of nucleotides in accordance with the invention is characterized in that it comprises all or a portion of the concatenations shown in FIGS. 2, 3, 4, 5 or 6 and in particular the sequenced concatenations shown in these figures.

[0102] The invention also concerns fragments of said sequences and in particular the exons identified in the gene coding for the nAChR alpha6 subunit.

[0103] Further, the invention concerns recombinant cells comprising a nucleotide sequence as identified above or a vector containing it. Particularly advantageous recombinant cells are neuronal cells and in particular dopaminergic neurons or cells derived from the mesencephalic region of the brain of animals in accordance with the invention.

[0104] Other characteristics and properties of the invention will become apparent from the following examples.

KEY TO FIGURES

[0105]FIG. 1. Screening of the gene for the nAChR alpha6 subunit.

[0106] a) Genomic structure of the gene for the alpha6 subunit, including exons 1 to 6, and the structure of the transfer vector. Restriction sites: E, EcoRI; H, HindIII; S, SlaI; P, PstI; N, NotI. The NotI site shown in parentheses is in the phage used to construct the vector; it is absent from the wild type allele.

[0107] The 5 kb EcoRI-EcoRI and 4.6 kb HindIII-NotI fragments used to construct the homologous arms are shown above the wild type allele (WT). The recombination vector was used to produce replacement mutation; and it contains a neomycin resistance gene (Neo^(r)) as a positive selection marker and a diphtheria toxin gene (DTA) as a negative selection marker. After homologous recombination, a 4 kb deletion including exons 1 and 2 had been formed. The DNA fragments, digested with PstI, detected by the SaII-SpeI probe, are shown by the dotted lines.

[0108] b) Southern blot analysis identifying homozygous knockout (−/−), heterozygous (+/−) and wild type (+/+) alpha6 type animals.

[0109]FIG. 2: A: Restriction map of the bacterial artificial chromosome designated a6BAC (CNCM I-2504, deposited Jun. 22, 2000). The bac contains about 130 kb of insert deriving from a mouse line 129sv DNA library. This insert (α6) corresponds to the locus of the nicotinic acetylcholine receptor (nAChR) alpha6 subunit from exon I to exon VI.

[0110] B: Sequence for 13 kb insert, oriented in the direction of transcription.

[0111]FIG. 3: A: Restriction map for plasmid a6HN (CNCM I-2500, deposited on June 22, 2000).

[0112] Plasmid a6HN is constituted by a KS-Bluescript vector and a 4.6 kb insert cloned into the HindIII, NotI sites, deriving from the gene for the alpha6 subunit of the mouse nicotinic acetylcholine receptor (DNA deriving from ES cells from the SV/D3 strain). This fragment contains exons 3 to 5 of the alpha6 subunit. (Direction of transcription: HindIII to NotI).

[0113] B: Sequence for 4.6 kb insert. Exons 3, 4 and 5 are in capital letters. “n” represents non sequenced bases.

[0114]FIG. 4: A: Restriction map for a6EH plasmid (CNCM I-2501, deposited on Jun. 22, 2000).

[0115] The a6EH plasmid is constituted by a vector, SK, and a 4 kb insert, cloned into the EcoRI-HindIII sites. This inert is an EcoRI-HindIII fragment of the gene for the alpha6 subunit of the mouse nicotinic acetylcholine receptor (DNA deriving from ES cells from the 129 SV/D3 strain). It contains the first two exons of the alpha6 subunit (Direction of transcription: EcoRI to HindIII).

[0116] B: Sequence for 4 kb insert. Exons 1 and 2 are in capital letters. “n” represents non sequenced bases.

[0117]FIG. 5: A: Restriction map for plasmid a6EE (CNCM 1-2502, deposited on Jun. 22, 2000).

[0118] Plasmid a6EE is constituted by a SK-Bluescript vector and a 5 kb insert cloned into the EcoRI site. This insert is an EcoRI-EcoRI fragment of the alpha6 subunit of the mouse nicotinic acetylcholine receptor (DNA deriving from ES cells from the 129 SV/D3 strain). It contains 5 kb of non-coding sequence immediately upstream of the ATG of alpha6. These 5 kb of promoter are oriented in the SacI-KpnI direction of the SK.

[0119] B: Sequence for 5 kb fragment of promoter for the nAChR alpha6 subunit. The restriction sites are in capital letters. The sequence (EcoRI-EcoRI) is oriented from 5′ to 3′ in the direction of transcription. “n” represents non sequenced bases.

[0120]FIG. 6: A: Restriction map for plasmid a6PP (CNCM I-2503, deposited on Jun. 22, 2000).

[0121] Plasmid a6PP is constituted by inserting a 4.5 kb PstI-PstI fragment for the alpha6 subunit of the mouse nicotinic acetylcholine receptor (DNA deriving from ES cells from the 129 SV/D3 strain) into the PstI site of the SK-Bluescript vector. This 4.5 kb sequence belongs to the a6 promoter region. The 3′ end of this fragment is 3.9 kb in the 5′ direction from the ATG of the α6 gene.

[0122] B: Sequence for 4.5 kb insert, oriented in the direction of transcription. “n” represents non sequenced bases.

[0123]FIG. 7: A: Restriction map for plasmid a6KO (CNCM 1-2550, deposited on Aug. 29, 2000) contained in E. coli bacteria from the DH5α strain. The plasmid is used for homologous recombination into the locus of the alpha6 subunit of the mouse nicotinic receptor. This plasmid is constituted by a SK-Bluescript vector and a 12 kb insert. This insert contains two homologous arms belonging to the mouse alpha6 gene sequence (4 kb and 5 kb) flanking a neomycin resistance cassette (3.3 kb) (Neo^(r)). A diphtheria toxin expression cassette (DTA) is also included (1.4 kb).

[0124]FIG. 8: Localisation of the different restriction fragments a6PP, a6EE, a6EH, a6HN.

[0125]FIG. 9: Sequence of alpha6 locus of the nAChR receptor alpha6 subunit. The exons are in capital letters.

[0126]FIG. 10: In situ hybridization using an oligonucleotide probe located in exon 6 of the a6 subunit. Note the expression in the substantia nigra and the ventral tegmental area (SN/VTA) and in the locus coeruleus (LC). In knockout animals, the signal disappears completely.

[0127]FIG. 11: Quantification of autoradiographs obtained from in situ hybridization against subunits α3, α4, α5, α7, β2 and β4 of the nicotinic receptor. Cx=cortex; Th=thalamus; MHb=medial habenula; IPN=interpeduncular nucleus; Hp=hippocampus.

[0128]FIG. 12: Labelling of retinogenicular projections using fluorescent cholera toxin. Coronal sections representing left (a, c) and right (b, d) geniculate nuclei of Wt (a, b) and a6−/− (c, d) animals. Both eyes were injected with choleric toxin coupled with 2 different fluorophores (red for left eye, green for right eye). Note the absence of superimposition of labels, characteristic of normal segregation (scale: 350 μm).

[0129]FIG. 13: Autoradiograph of binding of αCtxMII to brain sections from Wt and mutant animals. Note the presence of binding sites in 3 major anatomic systems: the visual system (dorsolateral geniculate nucleus (dlGN), ventrolateral geniculate nucleus (VLGN), superior colliculus (SC), olivary pretectal nucleus (OPN), optic tract (opt)), the mesolimbic dopaminergic system (substantia nigra (SN), ventral tegmental area (VTA), nucleus accumbens (Nacc), striatum (Str)) and the interpeduncular habenulo system (interpeduncular nucleus (IPN) and medial habenula (MHb)).

[0130]FIG. 14: Displacement of binding of epibatidin by αCtxMII on striatum membrane preparations.

[0131]FIG. 15: Release of dopamine induced by application of 3 μM nicotine to striatal synaptosomes in the absence (a) or presence (b) of 100 nM of αCtxMII. Dose-response curve for dopamine release in response to nicotine in α6−/− (c) or α4−/− (d) animals. The maximum response was normalized to 1. Note the loss of affinity of an order of magnitude in the α4−/− animals.

EXAMPLES

[0132] Materials and Methods

[0133] Preparation of “knockout” mice (mice mutated for the gene coding for the nAChR alpha6 subunit, expression in the functional form of the alpha6 subunit of nAChR receptor being altered).

[0134] The majority of protocols used to prepare the knockout mice are described in Laboratory protocols for conditional gene targeting . . . , Torres R. and Kühn R. Oxford University Press (1997).

[0135] 1.1. Construction of Recombination Vector

[0136] A strategy based on PCR (Israel, D. A PCR based method for high stringency screening of DNA libraries. Nucleic Acids Res., 1993, 21(11): p 2627-31) was used to screen a mouse genomic DNA library prepared from embryonic stem cells (ES cells) from the 129 SV/D3 line. Other commercially available mice (Janvier, Charles River) could be used. However, it is preferable to use the same line from which the ES cells used for homologous recombination are derived. Oligonucleotides were selected from the cDNA sequence of the rat alpha6 subunit. Forward primer: 5′ACTGCTGTGAAGAGATTTACACA3′ Reverse primer: 5′AAGATGGTCTTCACCCACTTG3′

[0137] This screening allowed a phage containing approximately 17 kb of the alpha6 subunit of the nicotine acetylcholine receptor (α6 nAChR) to be isolated.

[0138] Restriction mapping and partial sequencing enabled the position of the first five exons to be determined. The sixth exon was outside the cloned sequence (see FIG. 1). A recombination vector of the replacement type was then constructed to introduce a 4 kb deletion into the locus for the alpha6 subunit to remove exons 1 and 2. To prepare the recombination vector, the 4.6 kb HindIII-NotI fragment and the 5 kb EcoRI-EcoRI fragment were sub-cloned into KS-Bluescript plasmids to generate plasmids a6HN and a6EE (KS) respectively. To supply new restriction sites, the SpeI-XhoI fragment of the a6EE plasmid was then sub-cloned into a modified bluescript vector in which the polylinker had been replaced by SacI-SfiI-ApaI-SpeI-XhoI-XbaI-NotI to constitute the plasmid a6EE(NS). During this time, a Neo^(r) expression cassette was introduced into the XhoI-HindIII sites of a6HN. The XhoI-NotI fragment of the resulting plasmid was then sub-cloned into the SalI-NotI sites of a6EE(NS). Finally, a diphtheria toxin expression cassette was introduced into the SaII (of exon 5 in the alpha6 subunit)-NotI site of this plasmid, reducing the homologous length of the right arm to 2.5 kb.

[0139] 1.2. Transfection

[0140] 107 pluripotent embryonic stem cells (ES cells) derived from the 129sv/Pas mouse line (Kress et al., Non-permissiveness for mouse embryonic stem (ES) cell derivation circumvented by a single backcross to 129/Sv strain: establishment of ES cell lines bearing the Omd conditional lethal mutation, Mamm Genome, 1998, 9(12): p 998-1001) were electroporated with 20 μg of DNA of recombination vector linearized with ScaI using a Bio-Rad electroporation apparatus (250 V, 500 μF). After 8 days selection in a medium containing G418 antibiotic (Gibco-BRL) (400 μg/ml), 200 antibiotic-resistant colonies were removed and dissociated in trypsin for plating onto 96-well plates. After 2 days growth, each clone was dissociated in trypsin and plated onto two wells of a 48-well plate. The contents of one of the wells were used for freezing while the contents of the other were plated onto a 24 well plate to prepare the genomic DNA.

[0141] 1.3. Screening of Recombinant ES Cell Clones by Genomic Southern

[0142] a) Preparation of DNA

[0143] Wells in confluence in the 96-well plate were washed with PBS and incubated at 37° C. for 12 hours with 500 μl of cellular lysis buffer (100 mM tris-HCI pH 8: 5 mM EDTA; 0.2% SDS; 200 mM NaCl, 100 μg/ml proteinase K). The genomic DNA was then recovered by precipitation from ethanol and re-suspended in 150 μl of water.

[0144] b) Digestion and Transfer

[0145] 50 μl of this genomic DNA preparation was used for digestion with 50 units of PstI for 6 hours at 37° C. The digestion product was then charged onto 0.8% agarose gel and underwent TAE electrophoresis at 40 V for 20 hours. The digested DNA was transferred onto a nylon membrane (Hybon-N+, Amersham) by Southern transfer in a 20×SSC buffer. The DNA was fixed on the membrane by exposure to UV light for 5 minutes.

[0146] c) Labelling the Probe

[0147] 25 ng of the 800 bp SalI-SpeI fragment of the alpha6 gene was labeled using a random priming kit from Amersham (RPN 1607). This label was localized at the 3′ end with respect to the right homologous arm outside the screening construction.

[0148] d) Hybridization and Washing

[0149] Transferred membranes were pre-hybridized for 30 minutes in 40 ml of hybridization buffer (0.5 M NaHPO₄ (pH 7.6), 7% SDS, 0.1 mg/ml salmon sperm DNA) at 65° C. Hybridization was carried out in 10 ml of the same buffer containing 10⁶ cpm/ml of denatured probe (3 min at 100° C.) for 15-20 hours at 65° C. The membranes were then washed twice with 0.1SSC; 0.1% SDS and exposed in a Phosphorimager cassette (Molecular Dynamics) for 24 hours before revealing.

[0150] This probe (SaII-SpeI fragment) and this restriction enzyme (PstI) allowed two bands to be detected in the recombinant clones: an 8 kb band corresponding to the wild type allele and a 4 kb band corresponding to the mutated allele.

[0151] The three recombinant ES cell clones obtained were multiplied and kept frozen in small aliquots in liquid nitrogen before use in aggregation.

[0152] 1.4. Preparation of Chimeras

[0153] Chimeras were obtained using a modification of the aggregation protocol as described by Khillan J. and Y. Bao, Preparation of animals with a high degree of chimerism by one-step co-culture of embryonic stem cells and preimplantation embryos. Biotechniques 1997, 22(3), p 544-9). Culturing overnight in a standard cell culture incubator produced small quantities of recombinant ES cells to be aggregated with wild type preimplantation embryos. The aggregates that developed in the form of blastocytes were then reimplanted in surrogate mothers in a state of pseudopregnancy to prepare the chimeric animals.

[0154] a) Preparation of ES Cells

[0155] Two days prior to aggregation, a cryotube containing 5×10⁶ ES cells was used to seed a 50 mm dish containing culture medium. The following day, the cells were dissociated and deposited in different concentrations on a 6-well plate (more particularly, dilutions of ⅓, ⅙, {fraction (1/12)} and {fraction (1/24)} were deposited on the initial 50 mm plate to use in the three forthcoming aggregation days).

[0156] The day of aggregation, the ES cells underwent mild dissociation (4 min in trypsin) to generate small aggregates of ES cells (4-20). The trypsin was inhibited by adding serum-containing medium. After mild dissociation using a 10 ml pipette, the cells were spread on a gelatinized tissue culture plate and placed in an incubator for 20 minutes. This step allowed selective removal of nutritive fibroblasts and large aggregates of ES cells which sedimented more rapidly. The supernatant containing the ES cells was harvested and kept on ice while the embryos were prepared.

[0157] b) Embryo Preparation

[0158] Five days prior to aggregation, 3 week old female mice (CD1 or C57BI6) were superovulated by injecting 5 units of PMS followed 46 hours later by an injection of 5 units of HCG (Intervet) then mated with two-month old male mice from the corresponding line, i.e., CD1 or C57BI6. The day of aggregation (2.5 days pc), 9 superovulated mice were sacrificed to recover their embryos in the morula stage and which were preserved in M2 medium (Sigma).

[0159] The zona pellucida was then removed by incubation in Tyrode's solution (Sigma): this step was carried out at ambient temperature under a microscope. Once the zona pellucida had disappeared, the embryos were transferred into drops of M2 medium to limit their exposure to the Tyrode's solution.

[0160] c) Preparation of Aggregation Plates

[0161] 6 drops (5 μl) of aggregation medium (50% M16+50% standard ES cell medium) were placed on an untreated 35 mm culture plate. These drops were then covered with mineral oil to prevent evaporation. Using the end of dissection tweezers, 6-8 small depressions (aggregation wells) were prepared in each drop.

[0162] Each well had received a small aggregates (clumps) of ES cells (4-8 cells for C57BI6 embryos and 8-12 cells for CD1 embryos) and a morula. Care was taken to maximize contact between the cells and the embryo to allow aggregation. The plate was then placed in an incubator overnight.

[0163] d) Reimplantation of Aggregates

[0164] The day after aggregation, 90% of the aggregates had developed into blastocytes. These blastocytes were then reimplanted into the uterus of female B6D2 mice (2.5 pc) in a pseudopregnant condition.

[0165] 1.5. Preparation of Homozygous Knockout Mice

[0166] For each strain (C57BI6 or CD1), a germinal line transmitter was obtained and crossed with B6D2/F1 or CD1 mice (respectively) for selection by colour of the skin of the resulting newborns. Agouti-coloured newborns were then analysed by genomic Southern. The mutation had been transmitted to C57BI/6J mice by crossing for the subsequent experiments.

[0167] 2) Release of Dopamine from Superfused Striatal Synaptosomes

[0168] 2.1. Preparation of Synaptosomes

[0169] The striata from two mice were rapidly dissected onto ice (total moist weight=60 mg) and placed in 10 vol of 0.32 M sucrose/5 mM HEPES pH 7.5 cooled on ice. The striata were then homogenized in a glass/Teflon homogenizer (15 hits at 900 rpm). The homogenate was centrifuged at 1000 g for 5 minutes at 4° C. to remove the nuclei and cell debris. The supernatant was then centrifuged at 12000 g for 20 minutes at 4° C. The final residue, consisting of a crude synaptosomal preparation (P2), was resuspended in a superfusion buffer (6 mg of initial tissue weight/ml). The superfusion buffer (SB) was composed as follows: (mM): NaCl 128, KCl 2.4, CaCl₂ 3.2, KH₂PO₄ 1.2, MgSO₄ 0.6, HEPES (pH 7.5) 25, D-glucose 10, L-ascorbic acid 1 and pargyline 0.1.

[0170] 2.2. Charging Synaptosomes with Tritiated Dopamine

[0171] The synaptosome preparation was incubated with [³H] dopamine (100 nM) for 20 min at 37° C. then centrifuged at 12000 g for 5 minutes. The residue was resuspended in SB buffer and charged onto a 13 mm diameter A/E glass fibre filter (Gelman Sciences) in a light vacuum. (The striatal synaptosomes from 2 mice are generally sufficient for 12 filters).

[0172] 2.3. Superfusion

[0173] The superfusion apparatus used was commercially available (Brandel). It consisted of 6-20 identical channels. Each channel was constituted by a retention chamber (vol=100 μl) containing filters carrying the synaptosomes. This chamber was connected, via a Tygon tube (internal diameter=1.14 mm) to a multichannel peristaltic pump (Masterflex) and to an effluent collection device. The superfusion buffer (SB) was pumped continuously through the chamber at a rate of 500 μl/min.

[0174] After a 15 minute flushing period, the effluent was collected every minute in scintillation flasks containing 5 ml of scintillant. After collecting for 5 minutes, the drug being studied was administered for 1 minute and the effluent collected over a new 10 minute period. Finally, the quantity of tritiated dopamine released in each sample was measured using a scintillation counter (Wallac 1214 Rackbeta).

[0175] 2.4. Data Analysis

[0176] The basal level for dopamine release was estimated by adjusting the data from the first 5 samples collected prior to administration of the drug with an exponential decay curve (y=a.exp(-bt)). The release peak was then defined as the maximum of the ratio “100× (measured dopamine-base line)/base line” for the next 10 samples after administration of the drug. This calculation method avoided problems linked to differences in the total quantity of radioactivity present on each filter.

[0177] 3) Molecular and Anatomical Characterization of Knockout Mice for the α6 Subunit (α6−/−).

[0178] 3.1. Verification of Animals

[0179] A study of the expression of mRNA coding for alpha6 subunit of the nicotine acetylcholine receptor (nAChR) confirmed the results obtained in the rat and chicken (Le Novere et al., 1996, Eur J Neurosci 8, 2428-39; Vailati et al., 1999 Mol Pharmacol 56, 11-19). In wild type animals (Wt), a6 is expressed both in the ganglion cells of the retina and the catecholaminergic neurons (substantia nigra (SN), ventral tegmental area (VTA) and locus coeruleus (LC)). In α6−/− animals, however, we have shown the total disappearance of the mRNA of the α6 subunit, confirming the functionality of the deletion that was made (FIG. 10).

[0180] The inventors have also verified the absence of developmental compensation associated with any over-expression of other nAChR subunits to make up for the disappearance of α6 in mutant animals. In situ hybridization using oligonucleotide probes directed against the subunits α3, α4, α5, α7, β2, β4 have shown that such a mechanism did not exist in a6−/− animals, at least on a transcriptional level (FIG. 11).

[0181] On a general point, α6−/− animals are viable, present in Mendelien proportions in the litters, mature normally and do not exhibit any major physical or neurological defect.

[0182] 3.2 Absence of Anatomical Anomaly in the Nervous Systems of α6−/−

[0183] The inventors investigated the anatomical development of neuronal structures in which α6 is normally expressed.

[0184] a) The visual System

[0185] In the visual system, the inventors studied the ganglion cell projections of the retina in the dorso-lateral geniculate nucleus, since the anatomical organization of these projections is known to be regulated during development by the activity of nicotinic transmission in the retina. In contrast to that observed with β2−/− animals (Rossi et al., 2001 Proc. Natl. Acad. Sci. USA 98, 6453-6458), α6−/− mutants exhibited no alteration in the visual system at this level (FIG. 12).

[0186] b) The Dopaminergic System

[0187] In the dopaminergic system, the inventors have verified the absence of an anatomical anomaly, both as regards cellular bodies (in the mesencephalus) and in their projections in the striatum. They studied the expression profile of a number of dopaminergic markers the majority of which are known to be influenced by nicotine administration. These markers include the dopaminergic tyrosine hydroxylase enzymes (TH) and dopamine transporter (DAT), dopamine receptors D1 and D2 and neuropeptides (cholecystokinine (CCK), preproenkephaline (PPE) and preprotakikinine (PPT)). Once again, no difference was detected in the expression profile of these markers in the brain of α6−/− animals.

[0188] 4) Pharmacology of Nicotinic Receptors Containing the α6 Subunit

[0189] 4.1. Profile of Conventional Nicotinic Ligand Bonds:

[0190] The distribution of binding sites for a variety of nicotinic ligands in the brain of α6−/− animals was studied. While the [¹²⁵I]α-bungarotoxin bond was not modified in the mutants, the epibatidine, cytisine and nicotine bond has shown a reduction in the number of sites with high affinity for these ligands in the retinal projection region (superior colliculus (SC) and the dorso-lateral geniculate nucleus (dLGN)). In contrast, in all other structures, in particular the SN/VTA and the striatum, no other difference could be detected by comparison with the Wt animals. This result, which was somewhat surprising, can be explained by the abundance of type α4β2 nicotinic receptors in these regions which could have masked small variations in the total number of sites.

[0191] 4.2. Iodized α-Conotoxin MII Bond ([¹²⁵I]αCtxMII)

[0192] The most surprising result of this pharmacological study derives from a study of the αCtxMII bond. Indeed, this toxin was isolated and characterized as being specific for nAChRs containing a α3β2 interface (Cartier et al., 1996 J Biol Chem 271, 7522-8). However, the inventors have shown in α6−/− animals complete disappearance of all of the high affinity binding sites for this toxin (FIG. 13). Further, since other possible sites, termed low affinity sites, could not be detected by this autoradiographic technique, the displacement of the [³H]epibatidin bond by unlabelled αCtxMII on striatum membrane preparations was studied. Here again, the inventors have been able to show the total disappearance of sites with high and low affinity for αCtxMII (20% of the total number of epibatidine sites) in α6−/− animals. A further result arises from this study: while 20% of the binding sites for epibatidin were sensitive to αCtxMII and this sensitivity disappeared in the mutants, the inventors could not show a reduction in the total number of epibatidine sites in α6−/− animals. It appears that sites sensitive to αCtxMII were replaced in the mutants by an equivalent quantity of resistant sites (FIG. 14). The mechanism for this compensation remains unknown despite several hypotheses being postulated. In the light of the inventors' results, it is not very likely that the compensation mechanism is transcriptional in nature. In contrast (Klinik et al., 2001 J Neurosci 21, 1452-63) have suggested the existence of α4α6(β2)₃ type sites in dopaminergic neurons. In the absence of the α6 subunit, these sites would not disappear but would be replaced by (α4)₂(β2)₃ type sites which are insensitive to αCtxMII.

[0193] 5) The α6 Subunit Intervenes in Nicotinic Modulation of Dopaminergic Transmission.

[0194] 5.1. Effect of Nicotine on Dopamine Release

[0195] Despite these compensation mechanisms, the inventors studied dopamine release induced by nicotine in striatal synaptosome preparations. In a wild type animal, administering nicotine (3 μM) induced an increase in dopamine release (DA) of about 100% over the basal level. In α6−/− and α4−/− animals, this release peak was reduced to 50%. Further, in α4−/−×α6−/− double mutants, and in α2−/− animals, the effect of the nicotine was completely absent (FIG. 6a).

[0196] 5.2. Effect of αCtxMII on the Release of Dopamine Induced by Nicotine

[0197] The inventors also studied the interaction of αCtxMII with the release of DA induced by nicotine in α4−/−, α6−/− and Wt animals.

[0198] In Wt animals, αCtxMII (100 nM) reduced the DA release peak induced by nicotine to 50% above the basal level. In contrast, in the α6−/− animals, the release of DA induced by nicotine was completely insensitive to αCtxMII, while it was completely abolished in α4−/− animals (FIG. 15a, b).

[0199] These results suggest the presence of 2 types of receptors in the dopaminergic termini in the striatum, each contributing 50% of the nicotine effect. They are the α4β2 type receptors that are insensitive to αCtxMII and α6β2 type receptors blocked by this toxin. However, close examination of the dose-response curves in α4 and α6 knockout mice showed a change in the affinity of the response in α4−/− mutant animals (drop of one order of magnitude) (FIG. 15c, d). In the light of the results obtained with α6−/− animals, this can only be explained by a modification in the composition of the nicotinic receptors involved in the release of dopamine induced by nicotine. This led the inventors to modify their theory on the composition of the nicotinic receptors present on the dopaminergic termini in the striatum. They propose two types of receptors, (α4)₂(β2)₃ and α6α4(β2)₃ which would each contribute 50% of the nicotine response, and each having the same affinity for nicotine. In α6−/− animals, only the sub-type (α4)₂(β2)₃ would subsist inducing a response that was reduced by 50% insensitive to αCtxMII. In contrast, in α4−/− animals, the inventors suggest the formation of a novel sub type (α6)₂(β2)₃ that is sensitive to αCtxMII and with a lower affinity for nicotine (which would explain the change in the affinity of the nicotine response observed in α4−/− animals). The compensation mechanism described in α6−/− animals would also be present in α4−/− mutants. In contrast, the 50% reduction in the dopaminergic response on administration of nicotine to α6−/− animals indicates that the compensation observed in these animals is not functional.

[0200] 5.3. Study of cFos Expression in Response to Nicotine

[0201] The inventors studied the expression of “early” genes in response to nicotine in α6−/− animals. These genes are transcription factors that are expressed in cells in response to very diverse stimuli translating a “genetic” activation of the cell in which they are expressed. In the case of drug addiction, an increase in expression of one of these genes, cFos, has been observed in response to administration of morphine, cocaine or nicotine in the cells of the striatum. This effect, which is blocked by infusion of dopaminergic antagonists into the striatum, is assumed to be responsible for long term modifications observed during chronic drug taking (Nestler, 2001 Nat Rev Neurosci 2, 119-28). In α6−/− animals, preliminary results appear to indicate an absence of activation of cFos expression in response to nicotine. These results, however, have to be confirmed on a larger number of animals and extended to other early genes.

1 7 1 23 DNA Artificial Forward Primer 1 actgctgtga agagatttac aca 23 2 21 DNA Artificial Reverse primer 2 aagatggtct tcacccactt g 21 3 4494 DNA Artificial Plasmid 3 aagctttatg ggccaaggtg gtgagaaaag attcacagag gaggtgaaat ccaaatgatc 60 tttgatcaat attttaagtc acatgtcatt tctaagtcaa cagagcagag gcaactggaa 120 acgttcggga tttctgttac agttaaatag ctttcatgca gtctccagtc ttcatgtctg 180 acttcattag cacggataac ttagatttgt ctatttttag atataattct ctagttaaga 240 cttgtattag caagcacata gaagactgaa aaatattatt tcttccttcc aggatgaagt 300 caaccagatt atggagacca atctgtggct gcgtcacgta tgtgtccccc cctttgaatg 360 gcggcagaat gtatccactt agtgataaag ccacctgcat taactttttc gcaccccaac 420 ctatgataga taaagaatat ccttttcctt gctttctcct agtccttggg tcagctctgg 480 ttgcagttat attaatatag gcagcacatg ggcagagcct ggtgtctgac atggaaccct 540 ctggcctttc tcttttaagc tcccagttct ctttgtatca cttactgata ccaagnnnnn 600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnatctg gaaggactac agattgcgtt 1140 gggatccaac ggagtatgat ggcatcgaga cacttcgagt tccagcagac aacatctgga 1200 agcctgacat cgttctgtat aacnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnaatg ctgtcggcga cttccaggtc gaaggcaaga ccaaagctct tctcaagtat 2340 gacggtgtga taacctggac cccaccagcc atctttaaga gctcctgccc aatggacatc 2400 accttcttcc cgtttgacca tcaaaactgc tccctgaagt ttggttcctg gacttatgac 2460 aaggcagaaa tcgaccttct catcattggc tctaaagtag acatgaacga cttttgggaa 2520 aacagtgaat gggaaattgt cgacgcctct ggctataagc atgacatcaa gtacaactgc 2580 tgtgaagaga tttacacgga cataacctac tccttctaca tcaggaggtt gcccatgttt 2640 tacaccatca acctcatcat cccctgcctc ttcatttcct tcctcacggt gctggttttt 2700 taccttccct ccgactgtgg cgagaaagtg actctttgca tctccgttct gctttctctc 2760 actgtctttt tgctggtgat tacagagacc atcccatcca catctctcgt gatcccactg 2820 gtgggtgagt atctactgtt caccatgatc tttgtcacgc tgtccattgt ggtgaccgtg 2880 ttcgtgctga acatacacta caggacccca gcaacgcata ccatgcccaa gtgggtgaag 2940 accatcttcc ttcaggcctt cccctcgatt ctgatgatga ggaaacctct ggacaagaca 3000 aaggaggcag gaggtgttaa ggaccccaaa agccatacca agaggcctgc caaggtcaaa 3060 tttactcatc gaggagaatc caaacttcta aaggaatgcc accactgcca aaaatcaagt 3120 gacatagcac ctggaaagag aagatcaagc cagcagcctg cacggtgggt ggcagagaat 3180 tcagagcact cgtccgatgt tgaagatgtc atcgagagtg ttcaattcat agcagaaaac 3240 atgaagagcc acaatgaaac aaacgaggta aaagtggagc ccttttctcc agccagctgc 3300 acccctagca ggcctacagg cactttagag actagtcaga gcgtcagtgg gagttacata 3360 tgtggaacag tcagggaccg tcacctaaga ccagctctat tatcatgaag ccttgtggga 3420 cctgggttca agtttaggga gctatagtga gaggatatat gtagtcctac aacaaatctt 3480 cagcctgcat ttacttacgg tgaggtctag ccacagtgca catgcaggac aagccttcct 3540 caaggaacaa gcctccaatg catcgaacac tgacaaagtg agggtgggaa gggagactgt 3600 agaaatcatt attaataaaa tcccaccggc gggcttgcta cctgctctaa tggtttgtgt 3660 tcccaaatga aacacacaca cacacacaca cacacacaca cacacacaca cagtctttgt 3720 gttttaatat gccgtataca gcacaatagc tgggccactg cctaccctcc atgctgttac 3780 tatacctccc gccaacaatc cccaagttat tacttactaa ttctatgtgc tatcttggtt 3840 gcccctagac ccagttgggc agcctctggg ccatgttttc cccggctatt tcaagtggtg 3900 gccatgtctc tctgtcctgc agtcttctca ggcctagcat ctcacctctt cctccacact 3960 ctcccggcat ggcagggtct cacttctcct cctcctcctc ctcctcctcc accacctcct 4020 ttctcctccc tccctcctcc ctcccctctt tcccctcccc ccttccttcc cagcctgaga 4080 actcctaaaa tcccacctct ccctgtcctt tccagctttg gctgttggca gctttattta 4140 ccaataagaa ccaactgcgg gcaggttccc agaaactaca ggcagacagt cccacgaaaa 4200 cagttttacg tggaccataa ttagcattca taatacgtgc agctacagag acacccaagg 4260 aaaagaagaa ctgtggtact gcctgaagcc ccagtggtga aagatgcccc ttagcagtca 4320 gcagctccca tcacacttcc tctactagag ggggacccag acatgagtta aagacatctt 4380 gaagatgtgc tcagtatgca cacgtattcc gtgcctgtgt cccacactct aatgcctaga 4440 gcttggctcc tcctacccca agcaagtgct caaggatagg gagatcgcgg ccgc 4494 4 4006 DNA Artificial Plasmid 4 gaattcagag agcttttatc atgctgaaca gccgggacca gggaaacctg cactccggtt 60 tatgtctgtg gctatgtgga ttcctggctc tctttaaagg tgagttatta cggcttatat 120 cctagaatac aattggtagt gtgcatttcc tcttctttct catcttgtct ggatccaaat 180 aattagagga ctttaattag ttaggcttaa cctaacagat atgatttgga aagtaaggta 240 gagaaaacat tgaagtacag tataatattc atacttcatc tctccaacta aaatatgctt 300 ttccaattag catacaaaat catctgcttt tgtagccatg tatgctacca atatgcttca 360 tttacttacg gtaatttgtt agctgggact tattagcctg cgagtcctac caatgtgtgt 420 aattgtcctc acttaggaaa cctgtcttat tgtctactaa gagtgagctt tcctgtctta 480 aaagttggct cagacctggc tggtgcacac acacacacac acacacacac acacacacac 540 acacacacga acaggcacac atgcacacat ctcggatgag gtttcagggg gtgctgatgg 600 tgagctccag agagaggaaa cagtagcaac agccaagaga gctcactatc ttctgaaaca 660 atgttttcat cctggttagt tacacaggag agaatgacag nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn gcagtacagg 1860 ctgtgaatct gaagagcagc tcttccacag gctgtttgct cactacaacc gcttcatccg 1920 gccggtggag aatgtctccg atcccgtcac ggtgcatctt gaattggcaa tcacgcaact 1980 ggccaatgtg nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nactccctgg gctgagcata 2520 tacaaaccat cacaccatgt ctgccagttg attgcagctt ttccagaaac tagtgcattt 2580 gaaaccatga ttcaaaattg ttcacctggg tgtcaactaa tattttcccg ataaagaggt 2640 ctaggctaat ggttccaaca acgtggtgtg gtactgtgtg tctctggcct cctcctccat 2700 tagggctcac tgagctcagt gtccattgaa atggtctgga gttgcatgaa gactatgatt 2760 tttccagagt aggtcctgac tcatgacatt ttctaacacc tccaaggttg tgtattcccc 2820 atgaatacac accatatgga gcctatccaa accatggaag tgtctcaagg gaggcaggcc 2880 tgttaagaat ggtactgata attagtgtca actccctgaa ctcacccagg ttttgaagac 2940 tatgacaggt ttaggccagt tacctctaag acctcacgga agtgaggcag cagagcctat 3000 ccagctttga ccaacagcta gtacatactt acccagcatc tgttagaccc tacagattca 3060 aggcgactca ataccagagg gccctaatca atagagggga atcactggga agctgtgcag 3120 gctccatttt cctaagtctt tttgttgact ttaccaatcc cagggcctgt gttggctcca 3180 catgacccac tggcagggag aagcttnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3300 nnnnnnnnnn nnnnnnncct gcgtagaaac ctcaaccagg ctgggatcct tcactcacag 3360 ggacctcaga tgtcttggaa aatgaaattt tgaatcccct gctgactgct aagaacctac 3420 tattaatacc caagctattc tgctgtgacc tgtcacctgt cctgactctc cttatgtcac 3480 ttcagggact gcttggacag taactcttca aagaagcctc cttgactcgt atattaaata 3540 gatgtgtgca tacacgcagg aatcactcca cctctcttgt tatatcatga tgtgttttat 3600 gctggcgtac acatttatta ttagctctgt ctttccaacc acaacagcta agagcacatg 3660 aaggcaggag ctttcttcac tctgtaactt gagtggaact ctgtaacaca gtggtcactg 3720 ggtaagtagg aatatccctt gtgactgaag ggcccgtgag ctgggaaagg gaatacagag 3780 ggagtgagta cctggtttca actggcaaac ttgcatgtga actaaactgc tgtttcagtt 3840 tggatcccag ggcaaatgca cacagtacat cccaggcttc tttgaggaaa ggccatgggg 3900 atagataaaa acagtgagta cctgtcgcca cctacaggac tctcccctct gtcgtccctg 3960 aacctctgcc ctcctccctg ttacctttca tgcatctagg aagctt 4006 5 5023 DNA Artificial Plasmid 5 ctgcaggaat tcttaggcaa atggatggaa ctagaaaata tcatcctgag tgaggtaacc 60 caatcacaaa agaacataca tggtatgcac tcactgataa gtggatatta cccccaaagt 120 ttggaatatc caagatacaa ttcacagacc aaatgaagct caagaagaag gaagaacaaa 180 gtatggatac ttcagtcttt cttagaaggg ggaacaaaat actcatggga ggagatacag 240 agacaaagtg tggagcagag actgaaggaa aagccattca gagactgccc cagctgggga 300 tccatcccat atacagtcac caaacccaga cactattgtt gatgccaaca agtgcatgct 360 gacaggagtc tgacatagct gtctcctgag aggctctgcc agtgcctgac aaatatagag 420 gggatgctca cagccaacca ctggactgag cacagggtcc tcaatggagg agttagagaa 480 aggactgaag gaccagaaga ggtttgcagc cccataggag gaacaacaat atgaactagc 540 cagtatcccc agagctccca gggactaagc caccaaccaa agagtacata tgcaggaacc 600 catggctcca gctgcatatg tagcagagga tgtcacttct caatgggagg agaggccctt 660 ggtcctgaga aggctcaatg ccccagtgta ggggaatgcc aagacaggga agtgggagtg 720 tgtgggttgg tgagcagggg ggaggaggaa aggagtaggg ggtctttgga ggggaaacca 780 ggaaaggaga taacatttga aatgtaaatg aagaaaatat ctaatttttt aagaaagaaa 840 aaagacaatt acagacttag ctgtgcctgt agccactgtt ctcctagagt tcagagcctg 900 cctgtctggg accagacacc tctggctgag gtttcttgcc tggcattatg tgcagggtcc 960 ggctccattt tctcacccag ccctgccttc cacagctctg tgcacaggag aggatccaag 1020 gagaagggaa gtggaggtgg tggcgacagt gacccagttc agaaaggact ctgaggcaca 1080 ttcaatctat aaagtattca tggctctaaa agccttccaa ggggatccaa agcatttgct 1140 gagctgcagn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn naactaggaa 4320 tatttcagac agatgcctaa ctgctaaggc tcagagaaac agcaaattaa gtggatatcc 4380 tttatggatg actgtgattt ttttttccta taatggaagc caagtaaata agatgctatt 4440 atcccactaa aagcagcggt tctcaacctc tgggtctcca ccccttgagg tcaaatgaca 4500 cttttaccga gggtcatcta agacattgga aaacacagat atttacatta tgactcacaa 4560 ccgtagaaaa attacactta tgaagtagca acgaactaat tttatccttg aggaataggg 4620 ggggtcacca caacatgcag atctgtatta aagggttgca gcattaggaa ggtttagaac 4680 cactgcccta agagtaggca agactgcagg tctctcatgg gacttgcctg tttgcctagt 4740 gtctcagcct gcctctcttc ttccctgcac agaggtctag ggtgacagga catgtgacag 4800 ccagccgagt gaagcagcag ggttggcttc ccttgccctt cacatctgtt tcagcctggg 4860 atgcagggag cagttgcatg atgtcccgtg ttttcacttc acagaagatc tgtgtgagct 4920 tccaggagct tcccggggct ttcaatatct gattctggca gctgacagac agctgtgtct 4980 tccacagccc tccgtgtaga aatcggaatt cgatatcaag ctt 5023 6 4500 DNA Artificial Plasmid 6 ctgcagaagc agcattaaga ctgtgctcta aatccaactc ctactttcct tggaagagac 60 acaagaaaaa gaggcaaggt gacagggcac agacaaagca ctgtagctac agatgcagag 120 atctgaatta ggaaactcta agccaaggca cacctggaat catcaatagc tggaaaagac 180 aagaaagatt ttctgtcaga gcctacagga aagtctggct ttgctaacac ctgcattttg 240 ggccattagc ctcagagctc tgaaatgata atattttgtt gtatttaccc accaaatttg 300 tggtgattta ttatgaggtc cctaggaaat aaataggact tggtatagtt tttttttttt 360 ttttcatttc caatcacaaa gcaagaatta ctgcaatgga attaaatttt atttcacaga 420 attgctggtt ctagtacaat ggtaccgatg ctgaaattgt ttctacatca ggagacttac 480 agtttaggca catttatttg tatgttgaag accccagata attactttga aaaaagagaa 540 gtcaagaggc tggaatggag ttccagaatc cccagaatct tgtcttggaa accatttcca 600 ctacaggatg tccttcactg aagttgctga gtggtgccca tgcaggacta atgaatccac 660 tgaaagctgt gtggcttaac tggagannnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnngaa 3360 ttcttaggca aatggatgga actagaaaat atcatcctga gtgaggtaac ccaatcacaa 3420 aagaacatac atggtatgca ctcactgata agtggatatt acccccaaag tttggaatat 3480 ccaagataca attcacagac caaatgaagc tcaagaagaa ggaagaacaa agtatggata 3540 cttcagtctt tcttagaagg gggaacaaaa tactcatggg aggagataca gagacaaagt 3600 gtggagcaga gactgaagga aaagccattc agagactgcc ccagctgggg atccatccca 3660 tatacagtca ccaaacccag acactattgt tgatgccaac aagtgcatgc tgacaggagt 3720 ctgacatagc tgtctcctga gaggctctgc cagtgcctga caaatataga ggggatgctc 3780 acagccaacc actggactga gcacagggtc ctcaatggag gagttagaga aaggactgaa 3840 ggaccagaag aggtttgcag ccccatagga ggaacaacaa tatgaactag ccagtatccc 3900 cagagctccc agggactaag ccaccaacca aagagtacat atgcaggaac ccatggctcc 3960 agctgcatat gtagcagagg atgtcacttc tcaatgggag gagaggccct tggtcctgag 4020 aaggctcaat gccccagtgt aggggaatgc caagacaggg aagtgggagt gtgtgggttg 4080 gtgagcaggg gggaggagga aaggagtagg gggtctttgg aggggaaacc aggaaaggag 4140 ataacatttg aaatgtaaat gaagaaaata tctaattttt taagaaagaa aaaagacaat 4200 tacagactta gctgtgcctg tagccactgt tctcctagag ttcagagcct gcctgtctgg 4260 gaccagacac ctctggctga ggtttcttgc ctggcattat gtgcagggtc cggctccatt 4320 ttctcaccca gccctgcctt ccacagctct gtgcacagga gaggatccaa ggagaaggga 4380 agtggaggtg gtggcgacag tgacccagtt cagaaaggac tctgaggcac attcaatcta 4440 taaagtattc atggctctaa aagccttcca aggggatcca aagcatttgc tgagctgcag 4500 7 17758 DNA Mus musculus misc_feature (687)..(3357) n equals a, t, g, or c 7 ctgcagaagc agcattaaga ctgtgctcta aatccaactc ctactttcct tggaagagac 60 acaagaaaaa gaggcaaggt gacagggcac agacaaagca ctgtagctac agatgcagag 120 atctgaatta ggaaactcta agccaaggca cacctggaat catcaatagc tggaaaagac 180 aagaaagatt ttctgtcaga gcctacagga aagtctggct ttgctaacac ctgcattttg 240 ggccattagc ctcagagctc tgaaatgata atattttgtt gtatttaccc accaaatttg 300 tggtgattta ttatgaggtc cctaggaaat aaataggact tggtatagtt tttttttttt 360 ttttcatttc caatcacaaa gcaagaatta ctgcaatgga attaaatttt atttcacaga 420 attgctggtt ctagtacaat ggtaccgatg ctgaaattgt ttctacatca ggagacttac 480 agtttaggca catttatttg tatgttgaag accccagata attactttga aaaaagagaa 540 gtcaagaggc tggaatggag ttccagaatc cccagaatct tgtcttggaa accatttcca 600 ctacaggatg tccttcactg aagttgctga gtggtgccca tgcaggacta atgaatccac 660 tgaaagctgt gtggcttaac tggagannnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnngaa 3360 ttcttaggca aatggatgga actagaaaat atcatcctga gtgaggtaac ccaatcacaa 3420 aagaacatac atggtatgca ctcactgata agtggatatt acccccaaag tttggaatat 3480 ccaagataca attcacagac caaatgaagc tcaagaagaa ggaagaacaa agtatggata 3540 cttcagtctt tcttagaagg gggaacaaaa tactcatggg aggagataca gagacaaagt 3600 gtggagcaga gactgaagga aaagccattc agagactgcc ccagctgggg atccatccca 3660 tatacagtca ccaaacccag acactattgt tgatgccaac aagtgcatgc tgacaggagt 3720 ctgacatagc tgtctcctga gaggctctgc cagtgcctga caaatataga ggggatgctc 3780 acagccaacc actggactga gcacagggtc ctcaatggag gagttagaga aaggactgaa 3840 ggaccagaag aggtttgcag ccccatagga ggaacaacaa tatgaactag ccagtatccc 3900 cagagctccc agggactaag ccaccaacca aagagtacat atgcaggaac ccatggctcc 3960 agctgcatat gtagcagagg atgtcacttc tcaatgggag gagaggccct tggtcctgag 4020 aaggctcaat gccccagtgt aggggaatgc caagacaggg aagtgggagt gtgtgggttg 4080 gtgagcaggg gggaggagga aaggagtagg gggtctttgg aggggaaacc aggaaaggag 4140 ataacatttg aaatgtaaat gaagaaaata tctaattttt taagaaagaa aaaagacaat 4200 tacagactta gctgtgcctg tagccactgt tctcctagag ttcagagcct gcctgtctgg 4260 gaccagacac ctctggctga ggtttcttgc ctggcattat gtgcagggtc cggctccatt 4320 ttctcaccca gccctgcctt ccacagctct gtgcacagga gaggatccaa ggagaaggga 4380 agtggaggtg gtggcgacag tgacccagtt cagaaaggac tctgaggcac attcaatcta 4440 taaagtattc atggctctaa aagccttcca aggggatcca aagcatttgc tgagctgcag 4500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5340 nnnnnnnnnn nnnnnnaagc ttnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnngcatgcn nnnnnnnnnn 5880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6300 nnnnnnnnnn nnnnnnnnnn ccatggnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnaactagga atatttcaga 7680 cagatgccta actgctaagg ctcagagaaa cagcaaatta agtggatatc ctttatggat 7740 gactgtgatt tttttttcct ataatggaag ccaagtaaat aagatgctat tatcccacta 7800 aaagcagcgg ttctcaacct ctgggtctcc accccttgag gtcaaatgac acttttaccg 7860 agggtcatct aagacattgg aaaacacaga tatttacatt atgactcaca accgtagaaa 7920 aattacactt atgaagtagc aacgaactaa ttttatcctt gaggaatagg gggggtcacc 7980 acaacatgca gatctgtatt aaagggttgc agcattagga aggtttagaa ccactgccct 8040 aagagtaggc aagactgcag gtctctcatg ggacttgcct gtttgcctag tgtctcagcc 8100 tgcctctctt cttccctgca cagaggtcta gggtgacagg acatgtgaca gccagccgag 8160 tgaagcagca gggttggctt cccttgccct tcacatctgt ttcagcctgg gatgcaggga 8220 gcagttgcat gatgtcccgt gttttcactt cacagaagat ctgtgtgagc ttccaggagc 8280 ttcccggggc tttcaatatc tgattctggc agctgacaga cagctgtgtc ttccacagcc 8340 ctccgtgtag aaatcggaat tcagagagct tttatcatgc tgaacagccg ggaccaggga 8400 aacctgcact ccggtttatg tctgtggcta tgtggattcc tggctctctt taaaggtgag 8460 ttattacggc ttatatccta gaatacaatt ggtagtgtgc atttcctctt ctttctcatc 8520 ttgtctggat ccaaataatt agaggacttt aattagttag gcttaaccta acagatatga 8580 tttggaaagt aaggtagaga aaacattgaa gtacagtata atattcatac ttcatctctc 8640 caactaaaat atgcttttcc aattagcata caaaatcatc tgcttttgta gccatgtatg 8700 ctaccaatat gcttcattta cttacggtaa tttgttagct gggacttatt agcctgcgag 8760 tcctaccaat gtgtgtaatt gtcctcactt aggaaacctg tcttattgtc tactaagagt 8820 gagctttcct gtcttaaaag ttggctcaga cctggctggt gcacacacac acacacacac 8880 acacacacac acacacacac acacgaacag gcacacatgc acacatctcg gatgaggttt 8940 cagggggtgc tgatggtgag ctccagagag aggaaacagt agcaacagcc aagagagctc 9000 actatcttct gaaacaatgt tttcatcctg gttagttaca caggnnnnnn nnnnnnnnnn 9060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnatgca tnnnnnnnnn nnnnnnnnnn 9180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnncata tgnnnnnnnn nnnnnnnnnn 9480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10200 nnnnnngcag tacaggctgt gaatctgaag agcagctctt ccacaggctg tttgctcact 10260 acaaccgctt catccggccg gtggagaatg tctccgatcc cgtcacggtg catcttgaat 10320 tggcaatcac gcaactggcc aatgtgnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnntcta gannnnnnnn 10620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnact 10860 ccctgggctg agcatataca aaccatcaca ccatgtctgc cagttgattg cagcttttcc 10920 agaaactagt gcatttgaaa ccatgattca aaattgttca cctgggtgtc aactaatatt 10980 ttcccgataa agaggtctag gctaatggtt ccaacaacgt ggtgtggtac tgtgtgtctc 11040 tggcctcctc ctccattagg gctcactgag ctcagtgtcc attgaaatgg tctggagttg 11100 catgaagact atgatttttc cagagtaggt cctgactcat gacattttct aacacctcca 11160 aggttgtgta ttccccatga atacacacca tatggagcct atccaaacca tggaagtgtc 11220 tcaagggagg caggcctgtt aagaatggta ctgataatta gtgtcaactc cctgaactca 11280 cccaggtttt gaagactatg acaggtttag gccagttacc tctaagacct cacggaagtg 11340 aggcagcaga gcctatccag ctttgaccaa cagctagtac atacttaccc agcatctgtt 11400 agaccctaca gattcaaggc gactcaatac cagagggccc taatcaatag aggggaatca 11460 ctgggaagct gtgcaggctc cattttccta agtctttttg ttgactttac caatcccagg 11520 gcctgtgttg gctccacatg acccactggc agggagaagc ttnnnnnnnn nnnnnnnnnn 11580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnncctgcgt agaaacctca accaggctgg 11700 gatccttcac tcacagggac ctcagatgtc ttggaaaatg aaattttgaa tcccctgctg 11760 actgctaaga acctactatt aatacccaag ctattctgct gtgacctgtc acctgtcctg 11820 actctcctta tgtcacttca gggactgctt ggacagtaac tcttcaaaga agcctccttg 11880 actcgtatat taaatagatg tgtgcataca cgcaggaatc actccacctc tcttgttata 11940 tcatgatgtg ttttatgctg gcgtacacat ttattattag ctctgtcttt ccaaccacaa 12000 cagctaagag cacatgaagg caggagcttt cttcactctg taacttgagt ggaactctgt 12060 aacacagtgg tcactgggta agtaggaata tcccttgtga ctgaagggcc cgtgagctgg 12120 gaaagggaat acagagggag tgagtacctg gtttcaactg gcaaacttgc atgtgaacta 12180 aactgctgtt tcagtttgga tcccagggca aatgcacaca gtacatccca ggcttctttg 12240 aggaaaggcc atggggatag ataaaaacag tgagtacctg tcgccaccta caggactctc 12300 ccctctgtcg tccctgaacc tctgccctcc tccctgttac ctttcatgca tctaggaagc 12360 tttatgggcc aaggtggtga gaaaagattc acagaggagg tgaaatccaa atgatctttg 12420 atcaatattt taagtcacat gtcatttcta agtcaacaga gcagaggcaa ctggaaacgt 12480 tcgggatttc tgttacagtt aaatagcttt catgcagtct ccagtcttca tgtctgactt 12540 cattagcacg gataacttag atttgtctat ttttagatat aattctctag ttaagacttg 12600 tattagcaag cacatagaag actgaaaaat attatttctt ccttccagga tgaagtcaac 12660 cagattatgg agaccaatct gtggctgcgt cacgtatgtg tccccccctt tgaatggcgg 12720 cagaatgtat ccacttagtg ataaagccac ctgcattaac tttttcgcac cccaacctat 12780 gatagataaa gaatatcctt ttccttgctt tctcctagtc cttgggtcag ctctggttgc 12840 agttatatta atataggcag cacatgggca gagcctggtg tctgacatgg aaccctctgg 12900 cctttctctt ttaagctccc agttctcttt gtatcactta ctgataccaa gnnnnnnnnn 12960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn natctggaag gactacagat tgcgttggga 13500 tccaacggag tatgatggca tcgagacact tcgagttcca gcagacaaca tctggaagcc 13560 tgacatcgtt ctgtataacn nnnnnnnnat gcatnnnnnn nnnnnnnnnn nnnnnnnnnn 13620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13680 nnnnnnnnnn nntctagann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14220 nnnnnnnnnn nnnnnnggat ccnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14640 nnaatgctgt cggcgacttc caggtcgaag gcaagaccaa agctcttctc aagtatgacg 14700 gtgtgataac ctggacccca ccagccatct ttaagagctc ctgcccaatg gacatcacct 14760 tcttcccgtt tgaccatcaa aactgctccc tgaagtttgg ttcctggact tatgacaagg 14820 cagaaatcga ccttctcatc attggctcta aagtagacat gaacgacttt tgggaaaaca 14880 gtgaatggga aattgtcgac gcctctggct ataagcatga catcaagtac aactgctgtg 14940 aagagattta cacggacata acctactcct tctacatcag gaggttgccc atgttttaca 15000 ccatcaacct catcatcccc tgcctcttca tttccttcct cacggtgctg gttttttacc 15060 ttccctccga ctgtggcgag aaagtgactc tttgcatctc cgttctgctt tctctcactg 15120 tctttttgct ggtgattaca gagaccatcc catccacatc tctcgtgatc ccactggtgg 15180 gtgagtatct actgttcacc atgatctttg tcacgctgtc cattgtggtg accgtgttcg 15240 tgctgaacat acactacagg accccagcaa cgcataccat gcccaagtgg gtgaagacca 15300 tcttccttca ggccttcccc tcgattctga tgatgaggaa acctctggac aagacaaagg 15360 aggcaggagg tgttaaggac cccaaaagcc ataccaagag gcctgccaag gtcaaattta 15420 ctcatcgagg agaatccaaa cttctaaagg aatgccacca ctgccaaaaa tcaagtgaca 15480 tagcacctgg aaagagaaga tcaagccagc agcctgcacg gtgggtggca gagaattcag 15540 agcactcgtc cgatgttgaa gatgtcatcg agagtgttca attcatagca gaaaacatga 15600 agagccacaa tgaaacaaac gaggtaaaag tggagccctt ttctccagcc agctgcaccc 15660 ctagcaggcc tacaggcact ttagagacta gtcagagcgt cagtgggagt tacatatgtg 15720 gaacagtcag ggaccgtcac ctaagaccag ctctattatc atgaagcctt gtgggacctg 15780 ggttcaagtt tagggagcta tagtgagagg atatatgtag tcctacaaca aatcttcagc 15840 ctgcatttac ttacggtgag gtctagccac agtgcacatg caggacaagc cttcctcaag 15900 gaacaagcct ccaatgcatc gaacactgac aaagtgaggg tgggaaggga gactgtagaa 15960 atcattatta ataaaatccc accggcgggc ttgctacctg ctctaatggt ttgtgttccc 16020 aaatgaaaca cacacacaca cacacacaca cacacacaca cacacacagt ctttgtgttt 16080 taatatgccg tatacagcac aatagctggg ccactgccta ccctccatgc tgttactata 16140 cctcccgcca acaatcccca agttattact tactaattct atgtgctatc ttggttgccc 16200 ctagacccag ttgggcagcc tctgggccat gttttccccg gctatttcaa gtggtggcca 16260 tgtctctctg tcctgcagtc ttctcaggcc tagcatctca cctcttcctc cacactctcc 16320 cggcatggca gggtctcact tctcctcctc ctcctcctcc tcctccacca cctcctttct 16380 cctccctccc tcctccctcc cctctttccc ctcccccctt ccttcccagc ctgagaactc 16440 ctaaaatccc acctctccct gtcctttcca gctttggctg ttggcagctt tatttaccaa 16500 taagaaccaa ctgcgggcag gttcccagaa actacaggca gacagtccca cgaaaacagt 16560 tttacgtgga ccataattag cattcataat acgtgcagct acagagacac ccaaggaaaa 16620 gaagaactgt ggtactgcct gaagccccag tggtgaaaga tgccccttag cagtcagcag 16680 ctcccatcac acttcctcta ctagaggggg acccagacat gagttaaaga catcttgaag 16740 atgtgctcag tatgcacacg tattccgtgc ctgtgtccca cactctaatg cctagagctt 16800 ggctcctcct accccaagca agtgctcaag gatagggaga tcaccacttg actgaggttt 16860 cccgccaggt gtctggagat gcttcatact atagctccta acatctgtaa ctcataggga 16920 aagaatatca gaaccaccac caccccccaa aaaacacact gagagacact gagaatgcct 16980 gtcataacat gtcacaggag agcaatggcc agtgagctca cctcctggct ggcatctttt 17040 agaatgtacg gcaatacgtt acgttgattt atcannnnnn nnnnnnnnnn nnnnnnnnnn 17100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17220 nnnnnnnnnn nnnnnnnnct ctcacttgct tcacactcag attcccccca aacagccagg 17280 cacacgtgac aagagcttga ctctcaagca gatatattgt gtagagcgaa gaaaggaccc 17340 ttatcacagg caaagggtca caaaagtctt tgatacagtt catagcccag tgatgtacat 17400 ctgctctccc ttagaccatg agtcctacat aggacatgca ggcagtcagg caggcaaaca 17460 taatagcaat aattggtttt tttgaaagga ttggagaaaa tgctttggag aaaaaaaaaa 17520 gtattcataa aacatagcat gttttcttat agtcttggtt tttaaagcta gccttgatcc 17580 tttgtttgca tttcaggtag aagacgactg gaaatacatg gctatggtgg tggacagagt 17640 cttcctttgg gtatttataa ttgtctgtgt gtttggaact gtggggctat ttctgcagcc 17700 actgcttggg aacacaggaa actcttaatt ggtattgtcc ctccgagctc atcaagct 17758 

1. A non-human mammal carrying a mutation in the gene coding for the alpha6 subunit of the nicotinic acetylcholine receptor (nAChR), said mutation preventing expression in the mammal of said nAChR alpha6 subunit in a functional form.
 2. A non-human mammal according to claim 1, characterized in that the gene coding for the nAChR alpha6 subunit is mutated by deletion of all or a portion of its exons.
 3. A non-human mammal according to claim 2, characterized in that the gene coding for the nAChR alpha6 subunit is mutated by deletion of at least a portion of exon 1 and/or of exon 2, said deletion being sufficient to prevent binding of said nAChR alpha6 subunit with its agonists and/or antagonists in the nAChR alpha6 subunit expressed in the mammal.
 4. A non-human mammal according to claim 3, characterized in that the gene coding for the nAChR alpha6 subunit is mutated by deletion of exons 1 and
 2. 5. A non-human mammal according to any one of claims 1 to 4, characterized in that the mutation for the gene coding for the nAChR alpha6 subunit is homozygous and leads to an absence of expression of the alpha6 subunit, said mammal having an (α6−/−) phenotype.
 6. A non-human mammal according to any one of claims 1 to 4, characterized in that the mutation for the gene coding for the nAChR alpha6 subunit is homozygous, said mammal having a (α6^(Δ1-2/Δ1-2)) phenotype.
 7. A non-human mammal according to any one of claims 1 to 6, characterized in that the mutation in the gene coding for the nAChR alpha6 subunit is inducible.
 8. A non-human mammal according to any one of claims 1 to 7, characterized in that it is a mouse.
 9. A non-human mammal according to any one of claims 1 to 8, characterized in that its gene coding for the nAChR alpha6 subunit is mutated by homologous recombination Nationale de Cultures de Microorganismes, the National Collection of Microorganism Cultures] with accession number I-2550 on Aug. 29,
 2000. 10. A non-human mammal according to any one of claims 1 to 9, characterized in that the gene for the alpha6 subunit is mutated heterozygously.
 11. A cell culture obtained from cells of a non-human mammal according to any one of claims 1 to 10, characterized in that they are neuronal cells.
 12. A cell culture according to claim 11, characterized in that the cells are mesencephalic neurons.
 13. A preparation obtained from cells of a non-human mammal according to any one of claims 1 to 10, characterized in that they are striatal synaptosomes.
 14. Use of a non-human mammal according to any one of claims 1 to 10 or of a cell culture according to claim 11 or claim 12 or of a preparation according to claim 13, for the study of the effects of given substances on nicotinic receptors.
 15. Use according to claim 14, for the study of the effects of addictive drugs on nicotinic receptors.
 16. Use according to claim 14 or claim 15, for determining the effect of agonists of the nicotinic acetylcholine receptors (nAChR) on dopamine release.
 17. Use of a non-human mammal according to any one of claims 1 to 10 or of a cell culture according to claim 11 or claim 12 or of a preparation according to claim 13, in screening nAChR agonist or antagonist substances.
 18. Use of a non-human mammal according to any one of claims 1 to 10 or of a cell culture according to claim 11 or claim 12 or of a preparation according to claim 13, in screening agonist or antagonist substances of the nicotinic acetylcholine receptors of catecholaminergic or dopaminergic neurons.
 19. A method for screening substances that can interact with the nAChR alpha6 subunit selected from substances interacting with nAChRs, comprising: bringing the selected substance into contact with a non-human mammal according to any one of claims 1 to 10 or with a cell culture according to claim 11 or claim 12 or with a preparation according to claim 13, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors; detecting the ligand/receptor type interaction between said substances and said nAChR alpha6 subunit.
 20. A method for screening substances that can interact with the nAChR alpha6 subunit selected from substances interacting with nAChRs, comprising: bringing the selected substance into contact with a non-human mammal according to any one of claims 1 to 10 or with a cell culture according to claim 11 or claim 12 or with a preparation according to claim 13, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors; detecting the amount of dopamine released, said detection being implemented in the striatum when a non-human mammal is used.
 21. A method according to claim 20, characterized in that the test substances are nicotinic nAChR agonists or nAChR antagonists.
 22. A method according to claim 20 or claim 21, in which the test substances are selected from the group formed by nicotinic drugs that are selective as regards dopaminergic neurons.
 23. A method for identifying a substance with an affinity for the nAChR alpha6 subunit, which is selective as regards nicotinic acetylcholine receptors restricted to dopaminergic neurons, comprising: bringing the selected substance into contact with a non-human mammal according to any one of claims 1 to 10 or with a cell culture according to claim 11 or claim 12 or with a preparation according to claim 13, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors; detecting the selective ligand/receptor type interaction.
 24. A method for identifying a substance with an affinity for the nAChR alpha6 subunit, which is selective as regards nicotinic acetylcholine receptors restricted to dopaminergic neurons, comprising: bringing the selected substance into contact with a non-human mammal according to any one of claims 1 to 10 or with a cell culture according to claim 11 or claim 12 or with a preparation according to claim 13, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors; detecting the amount of dopamine released, said detection being carried out in the striatum when a non-human mammal is used.
 25. A method according to claim 24 for identifying substances having a selective affinity for nAChR receptors comprising an alpha6 subunit, comprising: a) bringing the test substance with a binding activity to nAChR receptors into contact with a non-human mammal according to any one of claims 1 to 10 or with a cell culture according to claim 11 or claim 12 or with a preparation according to claim 13, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors; b) detecting the released dopamine, said detection being implemented in the striatum when a non-human mammal is used; c) comparing the increase in the amount of dopamine released in the striatum in step b) with the amount of dopamine released in the striatum of the non-human mammal or cell cultures expressing the nAChR alpha6 subunit.
 26. A plasmid containing all or part of the gene coding for the mouse nicotinic acetylcholine receptor alpha6 subunit, selected from the following plasmids deposited at the CNCM: a6HN: CNCM N° I-2500 deposited on Jun. 22, 2000; a6EH: CNCM N° I-2501 deposited on Jun. 22, 2000; a6EE: CNCM N° I-2502 deposited on Jun. 22, 2000; a6PP: CNCM N° I-2503 deposited on Jun. 22, 2000; a6KO: CNCM N° I-2550 deposited on Aug. 29, 2000; or the bacterial artificial chromosome (BAC) designated a6BAC deposited at the CNCM with accession number I-2504 on Jun. 22,
 2000. 27. A nucleotide sequence, characterized in that it is a sequence of the gene coding for the nAChR alpha6 subunit contained in a plasmid selected from a6HN (CNCM, I-2500), a6EH (CNCM, I-2501), a6EE (CNCM, I-2502), a6PP (CNCM, I-2503), a6KO (CNCM, I-2550) or in the bacterial artificial chromosome a6BAC (CNCM, I-2504).
 28. A nucleotide sequence selected from the concatenations represented in sequences SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or any part of said concatenations corresponding to a sequenced fragment of the gene for the nAChR alpha6 subunit.
 29. Recombinant cells, characterized in that they comprise a nucleic acid sequence according to any one of claims 26 to
 28. 30. Recombinant cells according to claim 29, characterized in that they are neuronal cells from the dopaminergic neuron group.
 31. Recombinant cells according to claim 30, characterized in that they are derived from the mesencephalic region of the mouse brain.
 32. A method for screening substances that can interact with the nAChR alpha6 subunit, selected from substances interacting with nAChR receptors, comprising: bringing the selected substance into contact with a cell culture according to claim 11 or claim 12, under conditions that allow interaction between said substance and the nicotinic acetylcholine receptors; apply a stress to said cell culture; observing the neuroprotective effect of said substance on said culture towards said stress.
 33. Substances obtained from a screening method according to any one of clams 20 to 25 or
 32. 